Background. Isoflurane disrupts brain development of neonatal mice, but its mechanism is unclear. We explored whether isoflurane damaged developing hippocampi through FASL-FAS signaling pathway, which is a well-known pathway of apoptosis. Method. Wild type and FAS- or FASL-gene-knockout mice aged 7 days were exposed to either isoflurane or pure oxygen. We used western blotting to study expressions of caspase-3, FAS (CD95), and FAS ligand (FASL or CD95L) proteins, TUNEL staining to count apoptotic cells in hippocampus, and Morris water maze (MWM) to evaluate learning and memory. Result. Isoflurane increased expression of FAS and FASL proteins in wild type mice. Compared to isoflurane-treated FAS- and FASL-knockout mice, isoflurane-treated wild type mice had higher expression of caspase-3 and more TUNEL-positive hippocampal cells. Expression of caspase-3 in wild isoflurane group, wild control group, FAS/FASL-gene-knockout control group, and FAS/FASL-gene-knockout isoflurane group showed FAS or FASL gene knockout might attenuate increase of caspase-3 caused by isoflurane. MWM showed isoflurane treatment of wild type mice significantly prolonged escape latency and reduced platform crossing times compared with gene-knockout isoflurane-treated groups. Conclusion. Isoflurane induces apoptosis in developing hippocampi of wild type mice but not in FAS- and FASL-knockout mice and damages brain development through FASL-FAS signaling. Isoflurane is a commonly used clinical anesthetic because it is fast-acting, produces little irritability, and stabilizes hemodynamic status of patients. It is also a convenient inhaled anesthetic for children. General anesthesia with isoflurane for children, ranging from babies to adolescents, is a common practice in modern anesthesiology for surgical or procedural pain relief .Learning and memory ability depends on structures within the medial temporal lobe, including the hippocampal region  and the adjacent perirhinal, entorhinal, and parahippocampal cortices . The devThe FAS singling pathway is a common way to induce apoptosis in cells. The FAS receptor protein is also known as APO-1 or CD95. It belongs to the subgroup of the tumor necrosis factor receptor (TNF-R) family that can trigger apoptosis. Its physiological ligand, FASL (CD95L), is a member of the corresponding TNF cytokine family. FAS and FASL play critical roles in the immune system, in particular in the killing of pathogen-infected target cells and the death of no longer needed, potentially deleterious as well as autoreactive lymphocytes . FurtherTo begin investigating this issue, we treated primary cortical neurons with isoflurane and performed a microarray analysis. We found that isoflurane upregulated some genes coding apoptosis-related proteins and downregulated the genes coding proteins involved in learning and memory. Importantly, we found that FASL-FAS signaling was activated in primary cortical neurons by isoflurane, while the levels of FASL and FAS proteins were increased. Based on these preliminary findings, we further investigated the effects of isoflurane on the development of the intact hippocampus in neonatal mice through the FASL-FAS pathway, as well as how hippocampal learning is affected by isoflurane.lpr/NJU and B6Smn.C3-Faslgld/NJU C57 mice together with their mothers  were housed in clean cages and the room temperature was maintained at 24 ± 1°C, with a 12-h light-dark cycle. We used neonatal mice aged 7 days to examine apoptosis and mice aged 25 days to examine the learning and memory. We chose these two ages because previous studies demonstrated that 7-day-old mice exhibit robust anesthesia-induced cell death, with mortality lower than 20% during 6-h anesthetic exposure [The Central Laboratory and Center of Animal Experiments of the Eye, Ear, Nose and Throat Hospital at Fudan University provided most of the experimental procedures and protocols used in this study. All efforts were made to minimize the number of animals used and their suffering. Seven-day-old C57BL6/J mice together with their mothers  and 7-day-old B6.MRL-Fasexposure , and micWild type C57BL/6 mice, FAS-knockout mice, and FASL-knockout mice aged 7 days were randomly assigned to two groups, treated with either control pure oxygen or isoflurane. In total, there were six groups: wild type isoflurane group, wild type pure oxygen group (wild type control group), FAS-knockout pure oxygen group (FAS-knockout control group), FAS-knockout isoflurane group, FASL-knockout pure oxygen group (FASL-knockout control group), and FASL-knockout isoflurane group. Isoflurane groups received 1.5% isoflurane in 100% oxygen for 2 h per day for a total of 3 days. Control groups received only 100% oxygen for the same period as the isoflurane groups. Temperature and humidity were kept steady. Pups were fed by their mothers daily after anesthesia. At postnatal day (P) 9, mice were killed and the hippocampi were used to detect the apoptotic factor, caspase-3, by western blot. Mice that were not sacrificed at P9 were kept until P25 for the Morris water maze test. Anesthesia with 1.5% isoflurane for 2 h in neonatal mice did not cause significant changes in blood pressure and blood gas showed in previous study .Mice were killed and the brains were immediately harvested, and hippocampi were dissected for western blot analysis. The harvested hippocampus tissues were homogenized on ice using radioimmunoprecipitation assay  plus protease inhibitors (1 mM PMSF). The lysates were collected, centrifuged at 12,000 rpm for 5 min at 4°C, and quantified for total protein with the enhanced BCA protein assay kit .μm) were cut on a cryostat , mounted on coverslips, and stored at −80°C. Coronal brain sections were studied for apoptosis by TUNEL staining. The other mice were fed to 25 days of age to test their learning and memory ability through Morris water maze.Another group of pups were perfused transcardially with heparinized saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were then removed and postfixed overnight in the same fixative at 4°C for 24 h. Thereafter, brains were frozen at −20°C and stored at −80°C until use. Serial coronal sections (10 http://www.broad.mit.edu/gsea/). Gene sets are available from the Molecular Signatures Database . Briefly, normalized microarray expression data were ranked/ordered by differential expression. Normalized microarray expression data were expressed in log2 scale. Up- or downregulated genes were selected based on both a change in expression by 1.5 or −1.5 and statistical significance (P < 0.05) via Student's t-test. Default parameters were used. The phenotype was permutated 1,000 times. Heat maps of the expression data were generated by color-coding gene expression intensities. The color code shows higher or lower expression relative to the mean expression of that gene in all samples.Microarray data were obtained from publicly available Gene Expression Omnibus (GEO) databases. Dataset GSE2982 was chosen as it fulfilled the requirements for our study. To investigate FAS signaling pathways that were dysregulated in isoflurane-treated neurons, gene set enrichment analysis (GSEA) was performed, following the developer's protocol  overnight at 4°C and subsequently with secondary antibodies  in TBST plus 5% nonfat dry milk. The caspase-3 antibody was used to recognize the caspase-3 fragment (17–20 kDa), which results from cleavage at aspartate 175, and FL-caspase-3 (35–40 kDa). The antibody to the nontargeted protein β-actin (42 kDa) was used to control for loading differences in total protein amounts. Between steps, the blots were washed with TBST for 20 min. The blot was visualized using the Kodak image station . Each band in the blot represents an independent experiment. We averaged the results from 3–6 independent experiments.The hippocampi were harvested at the end of the anesthesia and were subjected to western blot analysis using the methods described by Kim et al. . Brieflyμm) were cut on a cryostat  and mounted on coverslips. Sections were permeabilized with proteinase K solution (20 g/mL) for 20 min. Terminal deoxynucleotidyl transferase (TdT) and dUTP  were added to the sections and incubated in a humidified chamber at 37°C for 2 h. The reaction was then stopped and followed by colorization with DAPI  for 10 min. Then, coverslips were mounted on glass slides with antifade mounting medium . Finally, the sections were analyzed under a light microscope  with 5x and 20x objective lenses and photographs of the sections were taken. An investigator who was blind to the experimental design counted the number of TUNEL-positive cells using Image LAS V4.1 .Mouse brains were removed and kept at 4°C in 4% paraformaldehyde. Serial coronal sections  that remained constant during the studies. Water was kept at 20°C and opacified with titanium dioxide throughout all training and testing. A video tracking system recorded the swimming motions of animals and the data were analyzed using motion-detection software for the MWM . After every trial, each rat was placed in a holding cage, under an infrared heat lamp, before returning to its regular cage.P25 mice were tested in the MWM 4 times per day for 5 days. Each mouse was placed in the pool to search for the platform at a random starting point. Mice that found the platform were allowed to stay on it for 15 s. The time taken to find the hidden platform was recorded as escape latency. If a mouse did not find the platform within a 90 s period, it was gently guided to the platform and allowed to stay on it for 15 s. The video tracking system recorded the swimming motions of the animals, and the data were analyzed using motion-detection software for the MWM . At the end of the reference training (P29), the platform was removed from the pool and the mouse was placed in the opposite quadrant. Each mouse was allowed to swim for 90 s, and the number of times the mouse swam across the platform area was recorded as platform crossing times. After every trial, each mouse was placed in a holding cage under a heat lamp for 1-2 min to dry before being returned to its regular cage.t-test was used to determine the difference between the anesthesia and control groups in levels of FAS, FASL, caspase-3, and TUNEL-positive cells. A two-way ANOVA with repeated measurements was used to analyze the difference of caspase-3 levels between the anesthesia and control groups. Interaction between time and group factors in a two-way ANOVA was used to analyze the difference of escape latency between mice in the control group and mice treated with anesthesia in the MWM. The Mann-Whitney U test was used to detect the difference of platform crossing times between the isoflurane treatment and control conditions. There were no missing data for the variables of MWM (escape latency and platform crossing time) during the data analysis. In all experiments, differences were considered statistically significant at P < 0.05. GraphPad Prism software version 5.01  was used to analyze the data.Data regarding biochemical changes and escape latency are expressed as means ± SD. The data for platform crossing time were not distributed normally and thus are expressed as medians and interquartile ranges. The number of samples varied from 5 to 10, and the samples were distributed normally with the exception of platform crossing time . A two-sample We first investigated the possible connection among isoflurane treatment, FASL-FAS signaling, and neurotoxicity. In primary cortical neurons, isoflurane upregulated the expression of genes like apoptosis-related genes and downregulated the expression of genes involved in learning and memory Figures . The actP < 0.05, n = 5).We determined the degree of apoptosis by detecting the expression of caspase-3 in the hippocampus of wild type neonatal mouse after isoflurane treatment. We also measured the activation of the FASL-FAS pathway by detecting the expression of FAS and FASL proteins in the hippocampus. After treating P7 mice with isoflurane for 2 h per day for 3 days, wild type neonatal mouse showed large increases in the level of FAS and FASL proteins compared to control treatment Figures . We quanlpr and B6Smn.C3-Faslgld, resp.) are mice that have a nonfunctional mutation in the FAS gene and FASL gene, respectively [P < 0.05. The result of two-way ANOVA suggested that loss of FAS attenuates the increase of caspase-3 induced by isoflurane  belongs to a subset of the TNF-R family that is involved in death transducing signals and can trigger apoptosis of neurons. Its physiological ligand, FASL (CD95L), is a member of the corresponding TNF cytokine family . FAS expphocytes . FAS-medphocytes . Isoflurphocytes –14. Our phocytes , 24.In conclusion, clinically relevant isoflurane anesthesia can damage the developing mouse hippocampus by FASL-FAS signaling. So we should avoid using isoflurane anesthesia in children in clinic to reduce the harm in developing brain. We may also avoid the damage caused by isoflurane to children by exploring the blocker of FASL-FAS signaling. Our study provides a new direction for clinical research in reducing isoflurane-induced damage in children's brains.


Role of Elspar® (l-asparaginase from Escherichia coli) in regulation of l-Asn and l-Gln has been confirmed by the other researchers through experimental studies. Therapeutic research against ALL remained elusive with the lack of information on molecular interactions of Elspar® with amino acid substrates. In the present study, using different docking tools binding cavities, key residues in binding and ligand binding mechanisms were identified. For the apo state enzyme and ligand bound state complexes, MD simulations were performed. Trajectory analysis for 30 ns run confirmed the kinship of l-Asn with l-asparaginase enzyme in the dynamic system with less stability in comparison to l-Gln docked complex. Overall findings strongly supported the bi-functional nature of the enzyme drug. A good number of conformational changes were observed with 1NNS structure due to ligand binding. Results of present study give much more information on structural and functional aspects of E. colil-asparaginase upon the interaction with its ligands which may be useful in designing effective therapeutics for ALL.Acute lymphocytic leukemia (ALL) is an outrageous disease worldwide. The online version of this article (doi:10.1007/s13205-015-0339-9) contains supplementary material, which is available to authorized users. Uncontrolled division of cells is defined as cancer. Cancer of white blood cells (WBC), characterized by excessive multiplication of malignant and immature WBC (lymphoblast) in bone marrow is referred to as acute lymphoblastic leukemia (ALL). Methods of treatment of acute leukemia include chemotherapy, radiation therapy, steroids, and intensive combined treatments including stem cell or bone marrow transplants. Chemotherapy is the most preferred way of treatment among the above. Various drugs employed for treatment of ALL include asparaginase, dexamethasone, prednisolone, vincristine, daunorubicin, cyclophosphamide, cytarabine, etoposide, thioguanine, mercaptopurine, hydrocortisone, methotrexate etc.l-asparaginase has evolved as an important enzyme in growing enzyme industry, owing to its potential use in the treatment of ALL and lymphosarcoma . The possible side effects reported with Elspar® include severe allergic reactions ; fever; pain, redness, or swelling at the injection site; symptoms of liver problems ; symptoms of pancreatitis ; neurological seizures and induction of anti-asparaginase antibodies that inactivate the anti-cancer enzyme  and l-Gln (C5H10N2O3) whose molecular masses are 132.12 and 146.14 g/mol were retrieved from Zinc database with ID numbers 1532556 and 1532526, respectively. Then they were subjected for energy minimization using the MMFF (Merck Molecular Force Field)   correlations to accumulate the calculations  and 1NNS—l-Gln (complex 2) docked complexes gained from molecular docking to ratify the stability for anti-cancer enzyme in apo state and bound state with the substrates in dynamic system. Generating both the l-Asn and l-Gln topologies using PRODRG server is the early step in MD simulations  simulation method is 4 and the grid spacing for FFT (Fourier spacing) is 0.16. In the neighbor searching method, the short-range neighbor list cutoff of 1 Å is taken commonly for electrostatic interactions and van der Waal interactions. The LINCS , N-terminus of 1NNS has two conserved threonine residues with catalytic role.As per the structural description provided by the depositors a total of nine α-helices  and fourteen β-sheets  are present in it. Based on l-Asn and l-Gln were docked into the catalytic site of 1NNS. Dock runs of ligands on the enzyme were performed using HEX 8.0.0 and PatchDock.Two ligand substrates namely l-Asn and l-Gln resulting in less free energy of −160.89 and −165.60 kJ/mol, respectively. Results obtained by HEX were validated by PatchDock webserver followed by refinement of obtained results with FireDock server. Outcomes strongly supported the previous results with a very good binding efficiency between receptor and ligands with the calculated global energy values of −22.72 and −21.46 kcal/mol for l-Asn and l-Gln, respectively,  by FireDock server. Enzyme made inter molecular hydrogen bonds (shown in red color) with ILE-182, ASN-184 with l-Asn ligand and on the other side generated same inter molecular hydrogen bonds with ARG-116, SER-120, and ASP-152 when l-Gln as the ligand  with nd Table . The pooRg). The g_rms based results with backbone atoms  describes overall spread of molecule and was calculated using g_gyrate tool of GROMACS. Continuous drifts in gyration radii for apo state enzyme in the range of nearly 2.9 and 3.15 nm describe the dynamic nature of 1NNS. Docked complexes are better in terms of conformational stability with constant and low Rg values authorizing their better folding Below is the link to the electronic supplementary material.


IHL was determined at baseline, 30 min post-exercise and 4 h post-exercise. Additionally ATP/Total P ratio was measured at baseline and 4 h post-exercise. Compared with baseline values we did not observe any statistically significant changes in IHL within 30 min post-exercise in neither the fasted nor the glucose-supplemented condition. However, IHL was elevated 4 h post-exercise compared with baseline in the fasted condition , an effect that was blunted by glucose supplementation . Acute exercise does not decrease liver fat in overweight middle-aged men. Moreover, IHL increased 4 h post-exercise in the fasted condition, an increase that was absent in the glucose-supplemented condition. These data suggest that a single bout of exercise may not be able to lower IHL.Elevated hepatic lipid content  increases the risk of metabolic complications. Although prolonged exercise training lowers IHL, it is unknown if acute exercise has the same effect. Furthermore, hepatic ATP content may be related to insulin resistance and IHL. We aimed to investigate if acute exercise leads to changes in IHL and whether this is accompanied by changes in hepatic ATP. Twenty-one men (age 54.8 ± 7.2 years, BMI 29.7 ± 2.2 kg/m No statistically significant decrease was found directly after exercise, irrespective of the dietary condition. Hence it was concluded that acute exercise does not lower liver fat in young healthy men. In the other two studies, one study including male subjects with an average age of 28.9 years old1H-MRS) was increased rather than decreases after a 2 hours exercise bout (50–60% of VO2 max), and it was speculated that the increase in IHL upon exercise was due to increased free fatty acid (FFA) availability during exercise. A clinically more relevant question, however, would be if acute exercise lowers liver fat in a middle-aged overweight subject population who are prone to the development of fatty liver or may already have elevated IHL (NAFLD), but such information is presently lacking.Regular exercise has beneficial effects on metabolic risk factors associated with type 2 diabetes13459101213As mentioned above, a potentially biasing complication when measuring IHL post-exercise is the increase in plasma FFA that goes along with exercise in the fasted state (originating from exercise-mediated increase in adipose tissue lipolysis)151H-MRS to determine IHL before and after exercise performed with and without glucose ingestion to suppress plasma FFA levels, as well as 4 h post exercise. 31P-MRS was employed before exercise and 4 h post exercise to determine hepatic ATP concentrations.Next to IHL, hepatic ATP concentrations, a measure for liver energy status, have also been suggested to be related to insulin resistance and hepatic lipid accumulationTwenty-one middle-aged overweight men participated in this study. The subject characteristics of the entire group are shown in No significant differences in energy expenditure between the glucose-fed and the fasted state were found at baseline, during exercise or post exercise see . During There was a significant time (p < 0.0001), treatment (p < 0.0001) and time*treatment interaction (p < 0.0001) effect for plasma FFA concentrations. Plasma FFA concentrations increased with time during exercise and recovery from exercise in the fasted state, with a significant higher plasma FFA concentration at the end of exercise (t = 120) and 4 hours post-exercise (t = 360) compared with before exercise (t = −60) (p < 0.0001), whereas plasma FFA concentrations decreased over time in the glucose-fed state (p = 0.002). Furthermore, plasma FFA concentrations were substantially higher at the end of 2 hours of exercise and 4 hours post-exercise in the fasted state (p < 0.0001) compared with the glucose-fed state .Plasma glucose concentrations showed a significant time (p < 0.0001), treatment (p < 0.0001) and time*treatment interaction (p < 0.0001) effect. Thus, plasma glucose concentrations increased with time during exercise and recovery from exercise in the glucose-supplemented state, with a significant higher plasma glucose concentration at the end of exercise (t = 120) and 4 hours post-exercise (t = 360) compared with before exercise (t = −60) (p = 0.0001), whereas plasma glucose concentrations decreased over time in the fasted state (p = 0.0001). Moreover, plasma glucose concentrations were higher at the end of 2 hours of exercise and 4 hours post-exercise in the glucose-supplemented state (p < 0.0001) compared with the fasted state .Plasma triglyceride concentrations showed a significant time (p < 0.0001) and time*treatment interaction (p < 0.0001) effect in the fasted condition . Plasma 1H-MRS, before, within 30 min after cessation of exercise and 4 h post-exercise. IHL did not change significantly with acute exercise neither in the fasted nor in the glucose-supplemented condition, with IHL after cessation of exercise (t = 120) being 8.3 ± 1.9% of the water resonance in the fasted condition (p = 0.154) and 8.4 ± 1.8% in the glucose-supplemented condition (p = 0.181). However, IHL was increased 4 h post-exercise in the fasted condition compared with before exercise ); this increase in IHL post-exercise was absent in the glucose-supplemented condition   .When dividing subjects with low (<5.6%) and high (>5.6%) liver fat content, similar effects are found, with no significant changes in IHL within 30 min after cessation of exercise in both conditions and slightly increased 4 h post-exercise in the fasted condition. Furthermore, the increase in IHL 4 h post-exercise was similar for both groups (p = 0.630)  nor the glucose-supplemented (p = 0.582) condition . HoweverRecent evidence suggests that prolonged exercise training might have a lowering effect on IHL345There is an increasing prevalence of obesity worldwide, and obesity is associated with excessive storage of lipids (triglycerides) not only in adipose tissue, but also in non-adipose tissues, such as skeletal muscle and liver. Excessive lipid accumulation in the liver is associated with insulin resistance and type 2 diabetes. Lifestyle interventions like exercise training revealed that hepatic fat can be lowered by prolonged exercise training, hence preventing complications associated with hepatic fat accumulation345de novo lipogenesisWhereas IHL did not significantly change within 30 min after cessation of exercise, it was slightly increased 4 h post-exercise in the fasted condition. This finding is consistent with recent reports in young, healthy subjects, in which one exercise session also lead to an increase in IHL after exercise1315Next to measuring the IHL content, ATP/Total P ratio was determined at baseline and 4 h post-exercise in a subgroup of eight subjects. Although, ATP/Total P ratio did not significantly change 4 h post-exercise in neither the fasted nor the glucose-supplemented condition, ATP/Total P ratio had a tendency to be decreased in six out of eight subjects 4 h post-exercise in the fasted condition. It has been suggested that hepatic ATP levels are decreased in conditions such as insulin resistance and type 2 diabetes mellitus18In summary, we did not observe a decrease in liver fat after acute exercise in overweight middle-aged men. Moreover, IHL was increased post-exercise in the fasted state, an effect that is most likely due to the exercise-induced increase in plasma FFA levels. These data suggest that acute exercise is not responsible for the exercise-lowering effects on liver fat, and that a single bout of exercise may not be able to lower IHL, not even in a population with elevated IHL.Twenty-one middle-aged overweight men with a wide range of liver fat content participated in this study. None of the subjects participated in competitive sports and subjects with unstable body weight (>3 kg change in preceding six months) were excluded from the study. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the institution's human research committee and written informed consent was obtained from all participants. All methods were carried out in accordance with the approved guidelines and regulations.+) at 7:00 am after an overnight fast. IHL was investigated by proton magnetic resonance spectroscopy (1H-MRS). After this, a Teflon cannula was inserted into an antecubital forearm vein for sampling of blood and subjects rested for 30 minutes. Immediately after drawing the first blood sample, subjects ingested either 1.4 g/kg body weight of glucose (dissolved in water to a 20% solution and flavoured with 1 ml lemon juice) or the same amount of water. After this, subjects started exercising (t = 0 minute) on a stationary bike at 50% of their pre-determined maximal power output (Wmax) for two hours. During exercise, blood samples were drawn and substrate oxidation was measured by indirect calorimetry  every 30 minutes  while heart rate was constantly measured. Immediately after cessation of exercise (t = 120 minute), subjects were transferred to the MR facility for a second 1H-MRS measurement. Importantly, due to the transfer time and the preparation steps of the 1H-MRS measurement itself, IHL could only be measured within 30 min after cessation of exercise. Subsequently, subjects bed-rested for three hours, followed by a third 1H-MRS scan four hours post exercise. Additionally, in a subgroup of eight subjects, ATP/Total P ratio was measured by phosphorous magnetic resonance spectroscopy (31P-MRS) at baseline as well as four hours post exercise. During the 4-hour post exercise period, blood samples were drawn and substrate oxidation was measured for 15 minutes every hour  in all subjects. The experimental design is depicted in Before the start of the study, body composition and maximal aerobic capacity were determined in all subjects. The experimental trial comprised two separate test days separated by at least one week and performed in random order. Subjects refrained from physical activity two days prior to the test days. Furthermore, subjects were instructed to consume a standardized meal the evening prior to the test days and stayed fasted from 10 pm onwards. On the test days, subjects reported to the laboratory    . The liver enzymes Gamma-GT, ASAT and ALAT were routinely measured during the screening visit and analysed in the clinical chemistry department in the hospital.in vivo by 1H-MRS. Measurements were performed on a whole body scanner  as reported previously3 Volume of Interest (VOI) was placed within the lower right hepatic lobe (point resolved spectroscopy (PRESS), repetition time (TR) = 4 s, Echo time (TE) = 23 ms, bandwidth (BW) = 1000 Hz, n = 1024 points, number of signal averages (NSA) = 64). To minimize motion artifacts, subjects were asked to breathe in the rhythm of the measurement and to be at end-expiration during acquisition of spectra. Water signal was suppressed using frequency-selective prepulses. Spectra without water suppression were acquired with identical settings (NSA = 16) and all spectra were fitted with AMARES2-corrected ratios of the CH2 peak, relative to the unsuppressed T2-corrected water resonance was calculated and converted to a tissue fat percentage (weight/weight) by assuming a water content of 71.1%, a CH2-proton density of triglycerides of 60.2 mol−1, a proton density of water of 2 mol−1, a molecular weight of triglycerides and water of 860 g/mol and 18 g/mol, respectively. Three subjects had to be excluded from the IHL analysis due to poor quality of spectra.IHL was determined In a subgroup of eight subjects ATP content was determined at baseline and four hours post exercise in both the fasted and the glucose-supplemented condition. Subjects were positioned in supine position with a 10-cm diameter transmit/receive surface coil positioned at the level of the liver. MRI scout images were acquired during a breath hold and one-dimensional spectroscopic imaging (SI) was performed with 8 phase-encoding steps and SI slice thickness of 30 mm . At least one slice was planned to be exclusively in liver tissue. To minimize motion artifacts, subjects were asked to breathe in the rhythm of the measurement and to be at end-expiration during acquisition of spectra. All spectra were fitted with the AMARES algorithmData are presented as mean ± SE. Hepatic lipid content was not normally distributed and therefore a non-parametric Friedman test was performed to test if there was an overall effect on IHL. Thereafter a pairwise comparison was performed with a Bonferroni correction for multiple comparisons. A two-way repeated measures ANOVA was performed to compare the mean differences between conditions for ATP, substrate oxidation and plasma values of FA, triglycerides, and glucose. All statistics were performed using SPSS 16.0  for Mac and p < 0.05 was considered statistically significant.31P-MRS protocol and performed acquisition, analysis and interpretation of data; M.K.C.H. has set up the study concept and design and was responsible for the draft of the manuscript and supervision of the study process; M.E.K. has set up the 31P-MRS protocol and performed analysis and interpretation of data; P.S. and V.B.S.H. have set up the study concept and design, interpreted the data, were responsible for the draft of the manuscript and supervision of the study process and obtained funding. All authors reviewed the manuscript.L.B. and B.B. have set up the study concept and design, performed acquisition, analysis and interpretation of data, were responsible for the draft of the manuscript and performed statistical analysis; P.A.E. has set up the


In this study, we investigate the effects of valerian root extracts (VE) on physical and psychological stress responses by utilizing a communication box.4) and 5-hydroxyindoleacetic acid (5-HIAA) were measured in the hippocampus and amygdala at 1 h after final stress condition, respectively.Eight-week-old ICR mice received oral administration of VE (100 mg/kg/0.5 ml) or equal volume of distilled water in every day for 3 weeks prior to being subjected to physical or psychological stress for 3 days, which are induced by communication box developed for physical electric shock and psychological stress by nociceptive stimulation-evoked responses. The stress condition was assessed by forced swimming test and serum corticosterone levels. In addition, norepinephrine (NE), serotonin (5-HT), and their metabolites such as 3-methoxy-4-hydroxyphenylethyleneglycol sulfate (MHPG-SO4 and 5-HIAA in the hippocampus and amygdala, respectively. The administration of VE significantly suppressed the increase of MHPG-SO4 and 5-HIAA in the two stress groups.Immobility time and corticosterone levels were significantly increased in both the physical and psychological stress groups compared to the control group. The administration of VE significantly reduced these parameters in both the physical and psychological stress groups. In addition, compared to the control group, physical and psychological stress groups showed significantly increased levels of MHPG-SOThese results suggest that VE can suppress physical and psychological stress responses by modulating the changes in 5-HT and NE turnover in the hippocampus and amygdala. Valeriana officinalis (VE) are popular herbal supplements and are widely used in the treatment of sleep disorders, anxiety, and epilepsy  and 3-methoxy-4-hydroxyphenylethyleneglycol sulfate [MHPG-SO4]) in the hippocampus and amygdala. Blood samples were obtained from each animal by cardiac puncture via the 1 ml syringe before obtaining the hippocampus and dentate gyrus. The samples were allowed to clot and were then centrifuged for 30 min at 1,000 g to separate out the serum. Corticosterone was measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit  following the manufacturer’s instructions. The absorbance was read at 450 nm. Brain was removed from braincase and the hippocampus and amygdala were separated on ice, and the samples were frozen using liquid nitrogen.Mice from all 5 groups  as described by Nadaoka et al. [g for 15 min. The supernatant was adjusted to pH 3.0 with 1 M sodium acetate and then passed through a 0.2-μm regenerated cellulose filter. An aliquot of this filter was injected onto a C18 reverse-phase column  in a HPLC system (Agilent 1100 series) equipped with an electrochemical detector. The mobile phase used with this aliquot (0.1 M acetate-citrate buffer with 17% methanol) allowed for the separation of the two major monoamines 5-HT and NE and their respective metabolites, 5-HIAA and MHPG-SO4[5-HT, NE, 5-HIAA, and MHPG-SOa et al. . The fro MHPG-SO4. Sodium The data represent the mean values for each experiment. To determine the effects of VE on PS and PCS, the differences between the means were statistically analyzed by using a one-way analysis of variance with Tukey’s post-hoc test.The immobility time of the PS-VE group was significantly decreased; it was 84.5% of that in the PS-V group. On the other hand, in the PCS-V group, immobility time was significantly increased to 125.6% of that in the control group. In the PCS-VE group, immobility time was significantly decreased compared to that in the PCS-V group  is a well-known screening tool for depressed animals , 31. DepIt was reported that a remarkable increase of plasma corticosterone level during and after both PS and PCS stress exposures , 34. Sim4 level has been considered to be more indicative of NE utilization in the brain [Next, we investigated the effects of VE on levels of NE, 5-HT, and their respective metabolites in the homogenates of the hippocampus and amygdala, which are regions most vulnerable to stress and the major targets for corticosterone, NE, and 5-HT –38. NE che brain –43. In ahe brain  and theshe brain .1A receptor and selective 5-HT reuptake inhibitors are clinically useful for treating various anxiety disorders [These results are supported by previous findings that PS causes a remarkable increase in NE turnover in various brain regions such as the cerebral cortex, midbrain, locus coeruleus, hypothalamus, amygdala, thalamus, and hippocampus, while PCS has been reported to cause an acute mild increase in NE turnover in the hypothalamus and amygdala . Agonistisorders , 48. 5-Hisorders , 50. AbnV. wallichii[The dissociation of 5-HT and 5-HIAA was supported by previous studies on stress response showing that increased brain levels of 5-HIAA without affecting 5-HT concentrations under stress condition –54. The wallichii.PS is induced by foot-shock stress, and PCS is generated by an exposure to the emotional responses caused by animal exposed to PS. PS and PCS animals significantly increase immobility time in forced swimming test, corticosterone levels in serum and turnover of 5-HT and NE in hippocampal and amygdala homogenates. PS dominantly modulates NE turnover, while PCS has a greater influence on 5-HT turnover. VE administration significantly suppresses the PS and PCS response by reducing the immobility time in forced swimming test, plasma levels of corticosterone and turnover of 5-HT and NE. These results suggest that VE could be ameliorated PS or PCS stress induced depression via control of plasma levels of corticosterone and turnover of 5-HT and NE.


Pinus sylvestris to N fertilization. The measurements were carried out in August 2013 on 90-year-old pine trees growing at Rosinedalsheden research site in northern Sweden. In spite of a nearly doubling of needle N content in response to the fertilization, no effect on the long-term shoot-scale C uptake was recorded. This lack of N-effect was due to strong light limitation of photosynthesis in all investigated canopy positions. The effect of greater N availability on needle photosynthetic capacities was also constrained by development of foliar phosphorus (P) deficiency following N addition. Thus, P deficiency and accumulation of N in arginine appeared to contribute toward lower shoot-scale nitrogen-use efficiency in the fertilized trees, thereby additionally constraining tree-scale responses to increasing N availability. On the whole our study suggests that the C uptake response of the studied N-poor boreal P. sylvestris stand to enhanced N availability is constrained by the efficiency with which the additional N is utilized. This efficiency, in turn, depends on the ability of the trees to use the greater N availability for additional light capture. For stands that have not reached canopy closure, increase in leaf area following N fertilization would be the most effective way for improving light capture and C uptake while for mature stands an increased leaf area may have a rather limited effect on light capture owing to increased self-shading. This raises the question if N limitation in boreal forests acts primarily by constraining growth of young stands while the commonly recorded increase in stem growth of mature stands following N addition is primarily the result of altered allocation and only to a limited extent the result of increased stand C-capture.Numerous studies have shown that temperate and boreal forests are limited by nitrogen (N) availability. However, few studies have provided a detailed account of how carbon (C) acquisition of such forests reacts to increasing N supply. We combined measurements of needle-scale biochemical photosynthetic capacities and continuous observations of shoot-scale photosynthetic performance from several canopy positions with simple mechanistic modeling to evaluate the photosynthetic responses of mature N-poor boreal  Q) within the canopy  to boreal, coniferous forests results in sharply increased basal area increment rates Tamm, . This wey Field, . Howevery Field, , 1996, ly Field, , constray Field, , and they Field, , 2016. WPinus sylvestris  to constrain leaf-scale photosynthetic capacity  increased shoot-scale photosynthesis, (2) lower shoot-scale NUE, (3) less optimal within-canopy N allocation pattern with respect to maximizing canopy-scale C gain due to N-binding to non-photosynthetic compounds and a greater limitation of photosynthesis by P deficiency in the upper canopy, and further that (4) canopy-scale C uptake may become substantially limited by P and arginine synthesis following N addition on N-poor boreal stands.We studied how needle-scale photosynthetic capacity and long-term shoot-scale photosynthetic performance in strongly N-limited P. sylvestris forest was regenerated from seed trees in the 1920's and the stand was approximately 90-years-old at the time of the current study. Here we use data from the 15 ha control and high-N plots, one of each, within the experiment. The ambient N deposition rate at the site is < 5 kg N ha−1 yr−1 , Mg (2.4%) and B (0.2%)  in August 2013. The experimental site was established for studies of the effects of N addition on stand-scale C cycling under N-poor boreal conditions. The homogeneous −1 (mean ± SD) in 2013, respectively. The mean tree height in 2013 was 17.5 ± 0.0 m (mean ± SD) at the control plot compared to 16.6 ± 0.2 m at the fertilized plot, and the leaf area index c. 2.7 on the control plot and c. 3.1 at the fertilized plot  were determined at three canopy levels in four dominant and co-dominant trees, growing within 10 m of each other. In the upper canopy measurements were made on current-year, 1-year-old and 2-year-old needles; in the middle and lower canopy only current-year and 1-year-old needles were measured. The A-Ci curves were determined from five attached fascicles (10 needles) using a LI-6400 Portable Photosynthesis System , with the standard 2 × 3 cm chamber and a light source (6400-02B LED Light Source), running in energy balance mode. To avoid mutual shading, the needles were separated and fixed in a horizontal plane at both ends using black PVC tape prior to insertion in the measurement chamber. Before each individual measurement series leaks were tested for by breathing around the chamber and monitoring the [CO2] inside it. The response of net photosynthesis (An) to photosynthetic photon flux density (Q) was measured after a period of dark acclimation for Q ranging from 0 to 1500 μmol m−2 s−1 with [CO2] set to 400 μmol mol−1. The CO2 response of An was measured immediately following the light response measurement for a [CO2] range from 60 to 2000 μmol mol−1 with Q set to 1500 μmol m−2 s−1. The A-Ci and the light response curves were determined at 20°C for all shoots.Needles were accessed using 16 m tall scaffolding towers located near the centers of the experimental plots. At each plot photosynthetic responses to intercellular COAn to Q in the 30–75 μmol m−2 s−1 range. The photosynthetic capacity parameters  were estimated from the A-Ci curves as described in Tarvainen et al.  was determined using the response of n et al. . Brieflyn et al. , includin et al.  was modii et al.  and the i et al. , 2003 we2 and H2O exchanges were measured continuously at each plot on two of the trees included in the photosynthetic capacity measurements. The measurements were made on 1-year-old attached shoots located at three canopy levels , near the shoots used for the capacity measurements. Shoot segments (55 mm) were inserted in temperature-controlled cuvettes modified for P. sylvestris (Image Q was measured with leveled quantum sensors  placed within 5 cm of the shoot. The cuvettes were constructed from transparent Plexiglas that transmitted c. 90% of the incident Q  running in open mode  were defined as mean An divided by N or P per projected needle area, Na or Pa (in g nutrient m−2 needle) respectively. Thus, LUE is expressed as mol CO2 mol−1 photons and NUE and PUE as mol CO2 g−1 N s−1 and mol CO2 g−1 P s−1, respectively.The continuously measured data were used to calculate the long-term light-use efficiency (LUE) defined as mean Nm). Needle total Kjeldahl P contents were determined using Seal AutoAnalyzer HR 3 . The foliar nutrient contents of P. sylvestris exhibit some seasonality  of the continuously measured needles, sampled later in the year, were expected to be greater than those of the needles used in the capacity measurements, although the two sets of needles likely had similar foliar nutrient contents in August. Furthermore, P. sylvestris needles tend to rotate around their axis and, thus, using whole needles for determining the leaf mass per projected needle area (LMA) results in greater estimates of LMA, Na, and Pa than analyses using shorter needle segments. This effect was quantified in a separate analysis that showed that LMA, Na, and Pa estimated for such segments were 12.5 ± 2.6% (n = 6) lower than for whole needles  and WinSEEDLE Pro 5.1a  analysis software. The needle segments (20 mm) used in capacity measurements were cut along the edges of the tape holding them in place, and the needles used in the continuous measurements were detached from the shoot axis prior to scanning. The needle dry mass was determined after drying to constant weight at 70°C and an elemental analyzer  was used to determine the mass-based needle N contents  system with a Waters Tunable UV detector  following the procedure described in Inselsbacher et al. . Needle The light climate of each shoot used in the photosynthetic capacity measurements is expressed as diffuse non-interceptance (openness) at the shoot location measured using LAI-2200 Plant Canopy Analyzer . The LAI-2200 was run in two sensor mode with one of the sensors placed above the canopy. The vertical variation in openness is shown in Supplementary material Figure .P. sylvestris trees with strongly differing foliar N contents growing in the field under otherwise comparable environmental conditions rather than to study treatment effects on the stand-scale per se.Because the overall experimental design consisted of non-replicated plots centered on eddy-covariance flux measurement towers and due to the need of scaffolding towers for crown access, the measured trees were not randomly selected. While this raises concerns regarding pseudo-replication  in needle photosynthetic, structural and chemical properties. Shapiro-Wilk's and Mauchly's tests were performed to test the data for normality and sphericity, respectively. When the sphericity assumption was met the factor effects were evaluated based on the multivariate tests (Wilks' lambda), and if this assumption was violated the degrees of freedom were corrected using Greenhouse-Geisser estimates of sphericity. In cases where the needle properties of 2-year-old needles were measured only in the upper canopy . A three-way repeated measures ANOVA (RMA) with needle age and canopy position as within-subjects factors and fertilizer treatment (plot) as a between-subjects factor was utilized to detect significant differences Pa, (iii)Na and Pa, (iv) area-based needle N content excluding arginine N (Nax), and (v)Nax and Pa. When both N and P were included , the co-dependencies of N and P were determined by multiple linear regression analysis that also included the N*P interaction term. Because fertilizer application was expected to affect the relationships between N, P and photosynthetic capacity parameters, the multiple regression analyses were carried out separately for each plot. Regression analyses were also used to investigate the between plots differences in the long-term shoot-scale photosynthetic performance responses to environmental variables and needle properties. The differences between the regression slopes and intercepts were evaluated using ANCOVA. If the relationship between the dependent and independent variables was non-linear data were log-log transformed prior to the ANCOVA analysis.The photosynthetic capacity responses to needle nutrient contents were evaluated by regression analyses based on: (i)An was modeled based on the approach by Farquhar et al.  and Ribulose-1,5-bisphosphate (RuBP) regeneration (Wj), limiting the gross carboxylation rate, the CO2 compensation point (Γ*), Ci and mitochondrial respiration (Rd):The shoot-scale r et al.  as descrr et al. , Medlyn r et al. , and Walr et al. . AccordiWc, is described as:The Rubisco-limited gross carboxylation rate, Kc and Ko are the Michelis-Menten coefficients of Rubisco for CO2 and O2, respectively, and Oi is the intercellular O2 concentration. The RuBP regeneration-limited gross carboxylation rate, Wj, is described as:where Q:where J is the rate of electron transport that is related to e is the apparent quantum yield of electron transport, assumed to be 0.24 mol electrons mol−1 photons , at 0°C, and its temperature response were determined from the continuous shoot-scale measurements made when Q was below 2 μmol m−2 s−1. The Rd value, thus, includes the respiration from the woody twig but does not account for the reduction in respiration rates in light . These cut-off Ci:Ca ratios were determined from the LI-6400 derived light response curves, with the lower boundary corresponding to the minimum Ci:Ca ratio at light saturation and the upper boundary to the maximum Ci:Ca ratio in darkness.The model was initially parameterized for 1-year-old needles utilizing plot specific data to test if model predictions of the long-term d Tables , 3, canoAn by foliar P deficiency and N binding to arginine, the model was run for the two continuously measured upper canopy shoots at the fertilized plot. In the first run the upper canopy mean Vcmax and Jmax from the capacity measurements on fertilized needles were used together with the continuously observed needle temperature, Q and Ci data. In the second run Vcmax and Jmax were estimated by extrapolating the Na dependencies of Vcmax and Jmax observed at the control plot . Trees growing at the fertilized plot exhibited significantly greater Na and Nm, as expected, but also greater Pm and lower P:N ratios than the trees at the control plot, while Pa did not vary significantly between the plots  of the shoots used in the photosynthetic capacity measurements suggest that the upper canopy needles received on average at least three times as much diffuse light than the lower canopy needles Table . The conVcmax and Jmax were significantly higher at the fertilized than at the control plot. Furthermore, no significant age-related or vertical within-canopy variations in Vcmax or Jmax were detected for the current-year and 1-year-old needles  of the needles did not vary significantly with N availability or age, but was higher in lower than mid-canopy Tables , 3. Boths Tables , 3. Howees Table . The JmaVcmax with both Na and Pa was detected for the 1-year-old needles . When data was divided into groups based on plot and age, no significant relationships were found between the capacity parameters and Pa . The Jmax of 1-year-old needles increased significantly with Na at the control plot  but not at the fertilized plot , while Vcmax of the 1-year-old needles did not vary significantly with Na at either plot . Expressing needle N content without arginine bound N did not improve the linear regression fits . Both Vcmax:N and Jmax:N were significantly greater at the control plot, while rate of return for P investment did not vary significantly between the plots .The predictions of the t Figure  but not t Figure . The imps Figure . At the s Figure . The accs Figure . AlthougQ measured in August 2013 (Table n = 2–3) precluded using analysis of variance  but the within-canopy variation was similar between the plots (P = 0.16). Shoot-scale LUE increased non-linearly with decreasing mean Q at both plots , or in LUE in general (P = 0.72), indicating that the long-term needle area-based photosynthetic performance between the plots was similar when light availability was accounted for. Because the light availability also was similar between the plots . Moreover, the daily integrated An scaled significantly  with daily integrated Q at both plots. Nitrogen-use efficiency increased significantly with light availability at both plots . Furthermore, the non-significant difference in the regression intercepts indicated that there was no treatment effect on shoot-scale NUE (P = 0.052). Like NUE, PUE increased significantly with mean Q at both plots  or the PUE responses to Q (P = 0.50).Shoot-incident s Figure . Overalls Figure . No signs Figure , the lony Figure  and simis Figure  and the s Figure . No signAn, it performed qualitatively well exhibiting a slope of 1.0 (for data pooled from both plots) when canopy average Vcmax, Jmax and shoot specific environmental drivers were used . The second model run estimated the potential limitation of upper canopy long-term An by P deficiency and N allocation to arginine. The non-limited capacity estimates were obtained by extrapolating the capacity-Na relationship observed at the control plot , suggesting a c. 10% mean reduction in long-term An for the two shoots due to capacity limitations. When the model was run using capacity estimates based on the observed upper canopy Na from the November harvest of the long-term measured shoots, 7.6 g N m−2 s−1, the predicted An was 0.43 ± 0.09 mol CO2 m−2 d−1, indicating a potential performance limitation of c. 13.5%.When the model was parameterized specifically for upper canopy shoots at the fertilized plot, the predictions were also reasonable being P. sylvestris to increased foliar N contents and their implications on the long-term stand-scale C cycling are discussed below.Boreal conifers are commonly considered N-limited , The Strategic Research Area “Biodiversity and Ecosystem Services in a Changing Climate” (BECC) and the research councils: The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning, The Swedish Research Council and The Swedish Governmental Agency for Innovation Systems.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study aims to compare the protein composition of high-density lipoprotein (HDL) particles in coronary heart disease (CHD) patients and controls by proteomic methods.HDL has been reported to exert pro-atherogenic properties in CHD patients. Accumulating evidence indicates that HDL composition, rather than the HDL-C level, determines its functions. The changes in HDL composition involved in the conversion of anti-atherogenic to pro-atherogenic properties in CHD patients are currently unknown.P<0.05).iTRAQ combined with nanoLC-MS/MS was performed to obtain a differential expression profile of the HDL pooled samples of the male age-matched CHD patients and controls (n = 10/group). Of the 196 proteins identified in the examined HDL, 12 were differentially expressed between the CHD patients and the controls, including five up-regulated proteins and seven down-regulated proteins. Using GO analysis, we determined that the up-regulated proteins were mostly involved in inflammatory reactions, displaying a potential pro-atherogenic profile. In contrast, the down-regulated proteins were mostly involved in lipid metabolism processes, displaying anti-atherogenic properties. To confirm the proteomic results, serum amyloid A (SAA) and apoC-I were selected and quantified by ELISA, in the same population as the proteomic analysis, as well as another independent population (n = 120/group). Consistent with the proteomic results, the amount of SAA was significantly increased, and apoC-I was significantly decreased in the HDL particles of CHD patients compared with those of controls (Our study shows that the HDL proteome changes to a pro-atherogenic profile in CHD patients, which might compromise the protective effects of HDL. Proteomic analysis of HDL composition may provide more relevant information regarding their functional properties than steady-state HDL-C levels. Coronary heart disease (CHD) is one of the leading causes of morbidity and mortality in China, as well as in the rest of the world, and early detection and diagnosis are the most effective measures for preventing the progression of the disease Inflammation plays a major role in changing the composition and function of circulating HDL To test the hypothesis that HDL particles are remodeled under inflammatory conditions in CHD without lipid regulating drugs, we compared the protein composition of HDL particles isolated from CHD patients with those isolated from controls, using the isobaric tag for relative and absolute quantification (iTRAQ) technique combined with nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). This is one of the most highly sensitive proteomic technologies, as it can detect and quantitatively analyze low abundance proteins in complex biological samples http://clinicaltrials.gov/ (Identification Number: NCT01543308).A total of 260 subjects were enrolled in this study, including 130 CHD patients and 130 healthy controls. All of the patients and controls were recruited from patients referred to the FuWai Hospital, Beijing, China, between July 2011 and June 2012. The diagnosis of CHD was defined according to WHO criteria as follows: significant stenosis ≥50% in at least one major coronary artery determined by percutaneous coronary angiography. Patients with acute myocardial infarction were excluded. Control subjects were determined to be free of CHD by percutaneous coronary angiography or computed tomographic angiography. Exclusion criteria included uncontrolled hypertension, triglycerides ≥5.0 mmol/L, severe obesity (BMI≥30), presence of thyroid, hepatic or renal disease, any chronic or acute infections, inflammatory illness, autoimmune disease and any type of cancers. In addition, subjects were excluded if they had regularly received lower lipid therapy in the previous two months, such as statins. All subjects were free of anti-inflammatory medications. The study protocol was approved by the Ethics Committee of the FuWai Hospital  and conducted according to the principles expressed in the Declaration of Helsinki. Written informed consents were obtained from all subjects before enrollment. This trial has been registered at In the initial discovery stage, iTRAQ combined with nanoLC-MS/MS was performed to obtain a global and a differential expression profile of the HDL pooled samples between the two groups in cohort 1, which included 10 male patients with CHD and 10 age- and sex-matched controls. Then, in the validation stage, target proteins were selected on the basis of the proteome maps and quantified by enzyme-linked immunosorbent assay (ELISA), in cohort 1 and an independent cohort 2 of 120 CHD patients and 120 controls. Blood samples were collected after overnight fasting and prior to percutaneous coronary angiography.Cholesterol, triglyceride, LDL-C and HDL-C concentrations were determined enzymatically using an autoanalyzer . HDL-C concentrations were determined after precipitation of apoB-containing lipoproteins by dextran sulfate with polyethylene glycol. LDL-C concentrations were calculated by the Friedewald equation Regarding the density cutoff of ultracentrifugation technique for HDL isolation, 1.063<d<1.24 g/ml and 1.063<d<1.21 g/ml are the two common reported cutoff values To enhance the detection and identification of medium- and low-abundance proteins for iTRAQ analysis, high-abundance proteins, such as albumin and IgG, were removed from the HDL samples using a ProteoExtrct Albumin/IgG Removal kit . The protein concentrations in high-abundance depleted HDL samples were determined using a bicinchoninic acid kit.Sample pooling has often been used in proteomics experiments, as it serves to reduce overall variability by minimizing individual heterogeneity Protein digestion and iTRAQ labeling were performed according to the manufacturer's protocol . Briefly, the proteins of the HDL pools from the two groups were precipitated using acetone, and then dissolved into 20 µl of dissolution buffer (AB Sciex USA). After dissolution, the protein content of each sample pool was quantified via Bradford assay. Eighty micrograms of the processed HDL were taken from each sample solution and digested with trypsin with a ratio of protein∶trypsin  =  30∶1 at 37°C for 16 hours. After trypsin digestion, peptides from the control and the CHD groups were labeled with iTRAQ tags 114 and 116 for 1 hour, respectively. The labeled peptide pools were then mixed and dried by vacuum centrifugation.2O, pH 10.0) and loaded onto a 4.6×250 mm Agela Venusil XBP C18 column containing 5-µm particles. The peptides were eluted at a flow rate of 0.7 ml/min with a gradient of 5–8% buffer B  for 1 min, 8–32% buffer B for 24 min, 32–95% buffer B for 2 min, 95% buffer B for 4 min, and 95–5% buffer B for 1 min. Elution was monitored by measuring the absorbance at 214 nm, and fractions were collected every minute. The eluted peptides were pooled into 8 fractions, and then vacuum-dried.HPLC was performed with a RIGOL L3220 HPLC pump system. The iTRAQ-labeled peptide mixtures were reconstituted with 4 ml of buffer A  coupled with an EASY-nanoLC-1000 system. The mobile phases consisted of solvent A  and solvent B . The HPLC fractionated peptides were dissolved in solvent A and loaded on a C18 trap column (100 µm×20 mm×5 µm). Chromatographic separation was performed with a C18 column (75 µm×150 mm×3 µm). The gradient was delivered at 450 nl/min and consisted of a linear gradient of mobile phase B initiating from solvent B, 3–90% over 78 min. The mass spectrometer was operated in positive ion mode with a resolution of 70 000 using a source temperature of 320°C. The applied electrospray voltage was 1.9 kV. For MS scans, the m/z scan range was 300 to 1400 Da.The MS/MS obtained spectra were searched against the NCBI reference sequence (RefSeq) database  using Proteome Discoverer software  with the MASCOT  search algorithm. The search parameters for tryptic cleavage and accuracy are built-in functions of the software. For protein identification and quantification, a peptide mass tolerance of 15 ppm was allowed for intact peptide masses and 20 mmu for fragment ions. Two missed cleavages were allowed in the trypsin digests. Carbamidomethylation of cysteine was considered as a fixed modification, and the conversions of N-terminal glutamine to pyroglutamic acid and methionine oxidation were considered variable modifications.http://david.abcc.ncifcrf.gov) and GO enrichment analysis was performed to identify GO terms that were significantly enriched in differentially expressed proteins.All identified peptides had an ion score above the Mascot peptide identity threshold  of 1%), and a target protein was considered identified if at least two such unique peptide matches were apparent for the protein. For protein-abundance ratios measured using iTRAQ (116/114), we set a 1.5-fold up-regulation and 0.75-fold down-regulation change as the threshold and a two-tailed p-value <0.05 to identify significant changes. Gene Ontology (GO) functional classifications were analyzed with DAVID software  and serum amyloid A2 (SAA2) expressions in HDL were examined by ELISA. SAA2 is a precursor and further processed into the mature and active form of SAA t-test or Mann-Whitney test, and the nominal data were compared using the Chi-square test. A multiple linear regression analysis was performed to determine the association between the expression of SAA and apoC-I in HDL fractions (as a dependent variable respectively), and the difference variables of plasma hs-CRP and a history of smoking between the two groups. A P value <0.05 was considered significant.All statistics were performed using SPSS version 18.0 . The descriptive data are presented as the mean ± SD, and the continuous and nominal variables are presented as frequencies respectively. The continuous data were compared using the Student's P = 0.013). All subjects in the two groups were non-diabetic. There were no statistical differences regarding medical treatment of antihypertensive agents or aspirin between the two groups.The clinical characteristics of the study subjects in the discovery stage are displayed in Using iTRAQ and 2D LC−MS/MS, we compared the two-pooled HDL samples from the CHD patients and the controls. To be identified as true-positive, proteins were required to contain two or more peptides with a 99% confidence score and a 1% local FDR in each of the two analyzed groups. A total of 196 high-confidence proteins, corresponding with the 1101 unique peptides, were identified in the HDL fractions isolated by ultracentrifugation. P-value ≤0.05) relative to the control samples. Among them, 5 proteins were up-regulated, and 7 were down-regulated  were consistent with well-established HDL-associated proteins including SAA2, C5, fibrinogen beta chain, apoC-I, apoC-II, hemoglobin subunit alphaThe biological processes and the molecular functions of all of the proteins identified in the HDL fractions were classified using a Gene Ontology (GO) classification system . More thThe GO functional analysis indicated that the inflammatory response, acute-phase response, defense response, and platelet activation were the most significant functional processes represented by the up-regulated proteins , whereas the lipid/cholesterol transport and metabolism were the most significant functional processes represented by the down-regulated proteins  .Two proteins (one up-regulated (SAA) and one down-regulated (apoC-I)) were selected to confirm the changes observed in the proteomic analysis. They were chosen on the basis of their significant functions of promoting cholesterol metabolism and their involvement in an inflammatory response in the progression of CHD, according to the GO functional analysis. SAA and apoC-I were quantified in individual HDL samples of the same cohort, using ELISA kits. The protein levels were normalized to total HDL protein.vs. 68.7±12.4 µg/mg of HDL, n = 10/group, P = 0.024)   , and HDL= 0.040) .vs. 48.6±154.5 µg/mg of HDL, P = 0.001)   and hs-CRP  in the total validation population, and not associated with any other factors. HDL-associated apoC-I was negative-independently associated with CHD  and HDL-C  in the total validation population and not related to any other factors. In the CHD groups, HDL-associated SAA was significantly higher in patients with severe stenosis (Gensini >26) compared with patients with mild-to-moderate stenosis (Gensini ≤26) (P = 0.009). Neither gender nor medications were shown to have an impact on HDL-associated SAA and/or apoC-I in either CHD or control groups   , and the= 0.033) . In the  P>0.05) .In this study, we tested the hypothesis that HDL fractions are altered during CHD, using a proteomic method, and we observed that HDL fractions transition to a pro-atherogenic profile in CHD patients. Of the differentially expressed proteins, the abundance of potential pro-atherogenic proteins was increased and the abundance of anti-atherogenic proteins was decreased. The expression levels of SAA and apoC-I were validated, confirming the reliability of our findings.In this iTRAQ with nanoLC-MS/MS approach, we greatly improved the HDL proteome coverage by identifying 196 proteins associated with HDL, including over 90% of the previously reported proteins Among the up-regulated proteins, HDL-associated SAA was confirmed to be higher in CHD patients compared to controls. To our knowledge, this is the first study to quantitatively detect a significant elevation of SAA in HDL fractions of CHD patients, i.e. not only in plasma. Moreover, the HDL-associated SAA was found to be independently associated with plasma hs-CRP, an inflammatory marker, and thus linked the inflammatory conditions to HDL protein composition. SAA is an acute-phase protein with numerous pro-inflammatory actions and its concentration increases up to 1000 times in the blood during inflammatory responses et al. reported that HDL from acute myocardial infarction (AMI) patients expressed an inflammatory profile compared to controls, but not in the stable CHD patients Different from our observations, a small sample study (n = 10/group) by Alwali For the down-regulated proteins, lipid/cholesterol transport and metabolism were the most involved functional processes. Firstly, heart-type fatty acid-binding protein is believed to play a role in the intracellular transport of long-chain fatty acids that could decrease the risk of cardiovascular disease via the favorable effects on anti-inflammation vs. 144.6±17.0 g/dl, P<0.05) We also identified down-regulated hemoglobin in CHD patients. HDL-associated hemoglobin is thought to contribute to the pro-inflammatory nature of HDL in CHD patients, and HDL from CHD patients has been demonstrated to contain more hemoglobin One potential limitation is the use of ultracentrifugation for HDL isolation, which might strip off proteins during the procedure, possibly resulting in an under-estimation of the HDL composition. However, ultracentrifugation has been well established as a method of HDL isolation and is widely used to date In conclusion, the HDL proteome displays a pro-atherogenic profile that might compromise the protective effects of HDL in CHD patients. Enrichment of SAA has a potential relevance to the pro-inflammatory properties of HDL. Recent studies have stressed that the simple concentration of HDL-C may not directly reflect HDL function Table S1The two density cutoff values of ultracentrifugation technique for HDL isolation.(XLSX)Click here for additional data file.Table S2The purity of 20 random HDL samples.(XLS)Click here for additional data file.Table S3Protein summary report identified in HDL samples by iTRAQ.(XLSX)Click here for additional data file.


Background: Canonical inhibitors interact with cognate proteases through a protease-binding loop.Results: Protease-binding loop of corn Hageman factor inhibitor (CHFI) does not inhibit its cognate enzyme-activated coagulation factor XII (FXIIa).Conclusion: Nonloop regions of CHFI are required for specific inhibition of FXIIa.Significance: CHFI is the first canonical inhibitor whose protease-binding loop is not sufficient for cognate enzyme inhibition. iK = 3.2 ± 0.4 nm). CHFI-123, which lacks 34 amino acid residues at the C terminus and the fourth and fifth disulfide bridges, inhibited FXIIa with a iK of 116 ± 16 nm. To exclude interactions outside the FXIIa active site, a synthetic cyclic peptide was tested. The peptide contained residues 20–45 (Protein Data Bank code 1BEA), and a C29D substitution was included to avoid unwanted disulfide bond formation between unpaired cysteines. Surprisingly, the isolated protease-binding loop failed to inhibit FXIIa but retained partial inhibition of trypsin (iK = 11.7 ± 1.2 μm) and activated factor XI (iK = 94 ± 11 μm). Full-length CHFI inhibited trypsin with a iK of 1.3 ± 0.2 nm and activated factor XI with a iK of 5.4 ± 0.2 μm. Our results suggest that the protease-binding loop is not sufficient for the interaction between FXIIa and CHFI; other regions of the inhibitor also contribute to specific inhibition.Activated factor XII (FXIIa) is selectively inhibited by corn Hageman factor inhibitor (CHFI) among other plasma proteases. CHFI is considered a canonical serine protease inhibitor that interacts with FXIIa through its protease-binding loop. Here we examined whether the protease-binding loop alone is sufficient for the selective inhibition of serine proteases or whether other regions of a canonical inhibitor are involved. Six CHFI mutants lacking different N- and C-terminal portions were generated. CHFI-234, which lacks the first and fifth disulfide bonds and 11 and 19 amino acid residues at the N and C termini, respectively, exhibited no significant changes in FXIIa inhibition ( This bond Leu35 , 3. CHFInd Leu35 , and is sorders 58. Moreovsorders 5 and ischsorders 5, 11 modeThus, Factor XIIa represents a promising target for antithrombotic agents because it plays no significant role in hemostasis but is involved in pathological clot formation. This scenario provides the opportunity to develop novel antithrombotic drugs and nonthrombogenic implantable medical devices  based oni.e. one-chain) and modified (i.e. two-chain) forms of the inhibitor are active   . Thus, aAlthough the structure of the CHFI-FXIIa complex is not available, evidence suggests that CHFI is a canonical inhibitor. Both the uncleaved one-chain and cleaved two-chain forms of CHFI are reported to inhibit trypsin , 28 and Escherichia coli.Based on the available data related to small peptide serine protease inhibitors, we propose that the isolated protease-binding loop of CHFI is a promising primary structure for the development of new FXIIa inhibitors. In this study, we tested the inhibitory activity of a synthetic peptide that resembles the CHFI protease-binding loop and five recombinant truncation mutants of CHFI. Surprisingly, the cyclic peptide CHFI-2, which represents the CHFI protease-binding loop bridged with one disulfide bond, is unable to inhibit FXIIa but retains its inhibitory activity against bovine pancreatic trypsin and activated coagulation factor XI (FXIa). Our results suggest that regions outside the protease-binding loop of CHFI are likely to contribute to its inhibitory potency toward FXIIa. We also report the first simple protocol for soluble expression of CHFI in E. coli Rosetta-Gami 2 DE3  was used. The expression vector pET-28a was also obtained from EMD Millipore. Recombinant CHFI and its fragments were expressed under the control of a T7 promoter and induced using isopropyl β-d-thiogalactopyranoside.The host strain E. coli was obtained from Eurogen . The control CHFI protein from Zea mays was obtained from Enzyme Research Laboratories .The pLA-TA plasmid containing a synthetic version of the CHFI gene with codon usage optimized for TGCGGATCCTCTGCTGGTACCAGCTG-3′ and 5′-TGCAAGCTTAGATCTGCTCGGCATGG-3′, respectively. Specific oligonucleotides were designed to perform PCR mutagenesis for each recombinant CHFI fragment from the pET28a/CHFI template. PCR fusion was achieved as previously described  were used to digest the obtained PCR products and the pET28a vector. T4-DNA ligase (New England Biolabs) was used to ligate the pET28a vector to each CHFI fragment gene.E. coli Rosetta-Gami 2 DE3 cells were transformed with the resulting vectors via heat shock transformation, as previously described  and purified on a Ni2+-agarose column (Qiagen) using a gravity flow format. The column was washed with a solution of 25 mm Tris-HCl, pH 8.0, 300 mm NaCl, and 50 mm imidazole. The samples were subsequently eluted in a solution of 25 mm Tris-HCl, pH 8.0, 300 mm NaCl, and 200 mm imidazole and further analyzed via SDS-PAGE. The concentrations of the purified proteins were measured by UV absorbance at 280 nm. The extinction coefficient for each protein was calculated using Vector NTI® Advance Software (Invitrogen).After overexpression of the CHFI mutant proteins, m Tris-HCl, pH 8.0, 250 mm NaCl, and 4 mm imidazole.The purified proteins were diluted to reduce the imidazole concentration and concentrated on 3-kDa Millipore filters. The final protein samples were obtained in a solution of 110 mi.e. vector, host, and purification procedure) was used as a negative control. Substrates S2302 (for XIIa), S2765 (for trypsin), and S2366 (for XIa) were obtained from Chromogenix . Factors XIIa and XIa were obtained from Enzyme Research Laboratories and Hematologic Technologies Inc. , respectively. Bovine pancreatic trypsin was obtained from Sigma-Aldrich.Chromogenic assays were performed according to the substrate manufacturer's instructions, with minor changes. Recombinant enhanced green fluorescent protein (EGFP) that was obtained under the same conditions  in 96-well flat-bottomed plates . The rate of substrate hydrolysis was recorded at 405 nm using a Sunrise microplate spectrophotometer . We used 1 nm FXIIa and 500 μm S2302 in the FXIIa assays, 200 pm FXIa and 500 μm S2366 in the FXIa assays, and 1 nm trypsin and 500 μm S2765 in the trypsin assays. To calculate the iK values for the interaction of the CHFI mutants with FXIIa, trypsin, and FXIa, the residual activity of each protease was determined in triplicate for at least six inhibitor concentrations.Activity assays for factor XIIa, factor XIa, and trypsin were performed at 37 °C in reaction buffer (110 mv) was plotted against the concentration of inhibitor on reversed coordinates to give 1/v (s × OD405−1), which is dependent on C (nm or μm). Dependence was approximated linearly to obtain parameters a (y intercept) and b (slope).The rate of substrate hydrolysis at 405 nm   code 1BE1BEA ) was usesoftware . Missingsoftware  was usedsoftware . Hydrogesupplemental Table S1. The pre-equilibrated systems were fully energy-minimized in 5,000 steps. This step was followed by a 50-ns MD run at 298 K with parameters similar to those used in the equilibrating runs. All atoms were allowed to move. These 50-ns trajectories were used to analyze the conformational changes. To obtain stable conformations of the CHFI variants for further protein-protein docking, after the MD run at 298 K, the systems were slowly cooled to 4 K, with a temperature reduction of 1 K/1 ns. The resulting structures of the CHFI variants are further referred to as “MD structures.” The MD simulations were performed using the Lomonosov Moscow State University supercomputer . The obtained model system parameters are provided in computer .2R0L , in the current study. We used the protein-protein docking ClusPro server . The BLAST tool  on the U2R0L . This st2R0L . To checo server , 43 to eo server , 45.2O9Q, resolution 1.70 Å, paper in press) and FXIa , the mutants were designed to lack different N- and/or C-terminal CHFI regions and/or disulfide bridges. CHFI-12345 is a full-size CHFI without any truncations. CHFI-2345 lacks 11 amino acids and the first disulfide bond at the N terminus, and Cys55 is replaced by Asp. This mutant is identical to a previously described shortened form of CHFI . After cloning into the pET28a vector, the constructs were transformed into the E. coli Rosetta-Gami 2 DE3 expression host.DNA sequences for CHFI-12345, CHFI-1234, CHFI-2345, CHFI-234, CHFI-1245, and CHFI-123 were amplified from the synthetic CHFI gene using primers with mismatches to introduce BamHI and HindIII restriction sites and a single amino acid substitution. An additional stop codon was introduced before the HindIII restriction site in mutants CHFI-12345 and CHFI-1234; therefore, these mutants carry only an N-terminal His6 tag 1C. Afteri.e. N- and/or C-terminal tag with molecular masses of 3.8 and 1.5 kDa, respectively) were calculated as: 17.5 kDa for CHFI-12345, 19 kDa for CHFI-1245, 15.2 kDa for CHFI-1234, 18 kDa for CHFI-2345, 16 kDa for CHFI-234, and 15.5 kDa for CHFI-123. The theoretical molecular mass of wild type CHFI was calculated as 13.6 kDa. These values are consistent with the SDS-PAGE results (Z. mays seeds and recombinant CHFI (CHFI-12345) is caused by the remaining N-terminal purification tag. The approximate yields of the individual mutants ranged from 2.5 to 75 mg of purified protein/liter of cell culture. Recombinant EGFP was also expressed for further use as a negative control in chromogenic assays to exclude possible unspecific chromogenic substrate cleavage by traces of proteases from E. coli.The proteins were partially expressed in soluble form (data not shown), and after one-step nickel affinity purification, the samples were run on SDS-PAGE . The the results . The molE. coli  or C terminus (i.e. CHFI-1234) also inhibit FXIIa with the same activity as wild-type CHFI. The truncation of one disulfide bond from both the N terminus and the C terminus (i.e. CHFI-234) slightly decreases the efficiency of the inhibitor  (A). The truncation of the fourth and fifth disulfide bonds (i.e. CHFI-123) dramatically reduces the ability of the mutant to inhibit FXIIa (iK = 116 ± 16 nm) compared with the wild-type protein . A full-size CHFI mutant lacking the third disulfide bond also exhibits reduced inhibitory activity against FXIIa  (B). Inhibition of FXIIa by the synthetic peptide CHFI-2, which represents the protease-binding loop, was not observed . Thus, the mutants retain wild-type inhibitory activity until the third and fourth disulfide bonds are deleted from the molecule, with a drastic decrease observed for CHFI-123.The function of recombinant CHFI is indistinguishable from that of wild-type CHFI (CHFI-ER) from corn seeds. This finding suggests that the remaining purification tags do not influence the inhibitory activity of CHFI against FXIIa. Shortened mutants with the truncation of only one disulfide bond at the N ( < 0.05) A. The tr < 0.05) B. InhibiD and E, and D and E, and 4C) from 1 ± 0.6 nm for CHFI-1234 to 2.9 ± 0.5 nm for CHFI-2345, as long as disulfide bridges 2, 3, and 4 are present. CHFI-1245 exhibited a dramatically increased iK (250 ± 70 nm), as did the synthetic peptide CHFI-2 (12 ± 2 μm). No inhibitory activity was observed for CHFI-123 at concentrations up to 75 μm (data not shown). Because CHFI-2 retains partial inhibitory activity, the isolated protease-binding loop might be in the active conformation.Because no FXIIa inhibition by CHFI-2 was observed, this peptide and the other mutants were assayed against trypsin , MD simulations were performed. A 50-ns MD simulation was followed by cooling of the system to obtain conformations of all three inhibitor variants stable in solution. These conformations might differ from the conformation of CHFI-12345 in the crystalline phase that was provided by the x-ray data. The conformation of the Arg34 side chain, which is a residue located at the scissile bond, is of particular interest.To investigate whether the mutations cause conformational changes in the isolated protease-binding loop (CHFI-2) and CHFI-1245 , Arg34 forms a stable salt bridge with mutated Asp29 (A), causing a change in the shape of the loop carrying Arg34 (B). In CHFI-2, the angle between two α-helixes is different from that observed in the initial x-ray structure (C). Because of this conformational change, the structure of the cyclic peptide is more rigid; as a result, Arg34 and Asp29 are unable to form a salt bridge because they are separated by a distance of 7 Å, which is sufficient to dampen their interaction. These differences might alter the interactions of the CHFI variants with the active sites of their cognate enzymes. Because of the observed difference in the conformation of the Arg34 side chain between the x-ray structure and the structure obtained after MD cooling, two forms of each inhibitor were used for protein-protein docking: the x-ray structures with minimization of modified parts and the stabilized geometries after 50-ns MD at 298 K and slow cooling of the system to 4 K. A detailed account of the molecular modeling results is presented in the supplemental materials.During the MD simulation of CHFI-12345, the C29D mutant CHFI-1245, and the synthetic cyclic peptide CHFI-2, no dramatic conformational changes were observed. After cooling the CHFI-12345 system, the conformation of the Arged Asp29 A, causinng Arg34 B. In CHFtructure C. BecausD). Docking of the tripeptide analog of the FXIIa-specific substrate S2302 provided a position in the model active site that was suitable for peptide bond cleavage (see SI). Thus, this FXIIa model was used for protein-protein docking without further improvements. A detailed description of the protein-protein docking results for the three inhibitor variants and their cognate enzymes is provided in the SI.Homology modeling of the FXIIa structure provided a model with a serine-histidine-aspartic acid triad located in a cleft on the protein surface in a suitable position for performing catalytic substrate cleavage D. Dockini.e. the C29D mutant and the truncated cyclic fragment) . This finding indicates that conformations obtained after cooling the system in the MD simulation (i.e. stable in solution) for the inhibitor variants are more suitable for inhibition of the target enzymes than the crystalline phase conformation provided by the x-ray data . The protein-protein docking results for these MD structures are consistent with the experimental observations of inhibitory activity described above. All three CHFI variants exhibit good binding with the trypsin active site in a position favorable for cleavage of the bond between Arg34 and Leu35, but the binding of the cyclic peptide is weaker than that of the full-length inhibitors. This finding is consistent with the data obtained from trypsin inhibition assay (D and E). The docked position of CHFI-12345 with FXIa places Arg34 in the active site, but the position is relatively far from the catalytic serine. In addition, the scoring energy of the binding is rather poor compared with the energies obtained for the complexes with trypsin. Taken together, these data could explain the experimentally measured 3-fold increase in inhibition constants. In the case of CHFI-1245, the observed position is rather far from the active site and is stabilized by several salt bridges (E). The existence of such an alternative binding site could explain the poor experimentally observed inhibitory activity of CHFI-1245 against FXIa (C). The interaction of the cyclic synthetic peptide CHFI-2 with FXIa is similar to the binding of CHFI-2 to trypsin. The complexes between the FXIIa model and CHFI-12345 and its C29D mutant were similar to those of trypsin. However, in the case of CHFI-2, the most favorable position is located far from the active site and generated primarily by hydrophobic contacts (F).In most cases, more favorable scoring energies of protein-inhibitor complexes were obtained for conformations of CHFI and its analogs that were provided by comparing the MD simulations to the x-ray structure and its modifications  ∼40 times higher than that of the parental protein (0.5 nm)  .i.e. the loop) is rich in proline (C and i.e. trypsin and FXIIa) and for FXIa, for which the inhibition is much weaker. MD simulation and protein-protein docking were also performed to predict possible interactions between the peptide and its cognate enzymes.CHFI-2 . Only slight alterations of the angle between two α-helixes in CHFI-2 were observed compared with the initial x-ray structure of the loop (C). These results do not support the initial assumption that the isolated loop would exhibit extra flexibility.The MD results suggest that the spatial structure of the cyclic peptide is quite rigid, and no dramatic conformational changes compared with the loop in the wild-type inhibitor are proposed  is ∼20 times higher than that of wild-type CHFI (5.4 ± 0.2 μm). CHFI-2 also retains its inhibitory activity against trypsin; however, the difference in iK between CHFI-2 and wild-type CHFI is greater . Taken together with the MD results, these data suggest that the isolated protease-binding loop is likely to be in an active conformation because it can inhibit trypsin and FXIa. The docking results support these experimental data, because the scissile bond of the peptide (i.e. Arg34–Leu35) is located in the active site of trypsin and FXIa.Our experimental results from the inhibition assay for the CHFI-2 peptide are consistent with previous findings , 21. TheB). Because the peptide is supposed to be in the active conformation, this lack of inhibition could be explained by an incorrect position of the scissile bond relative to the FXIIa catalytic site. We hypothesize that the functional position of the CHFI protease-binding loop in the cave of the FXIIa catalytic site is primarily determined by interactions between nonloop regions of CHFI and FXIIa. This hypothesis is supported by the CHFI-2-FXIIa docking results. The most favorable calculated position of the peptide is outside of the predicted active site on the FXIIa model structure (F). However, the disproportional loss of CHFI-2 inhibitory activity against FXIIa compared with trypsin and FXIa could also be partially caused by the elimination of the third disulfide bond and the other interactions between the loop and nonloop regions of CHFI.Surprisingly, no inhibition was observed when CHFI-2 was assayed against FXIIa B. Becaustructure F. HoweveiK = 50 ± 8 and 250 ± 20 nm, respectively) compared with wild-type CHFI . Unexpectedly, CHFI-1245 lost the ability to inhibit FXIa (iK = 490 ± 110 μm). This finding could be explained by the CHFI-1245-FXIa docking results, because an alternative CHFI-1245 binding site was present on the FXIa surface outside the active site (E). Nevertheless, the residual inhibitory activity of CHFI-1245 against FXIIa and trypsin suggests that although the third disulfide bridge contributes to the interaction of the CHFI loop with the FXIIa active site, the C29D substitution is not likely to be the only cause of the lack of activity of the CHFI-2 peptide against FXIIa.To better understand these possible reasons for the lack of activity of the CHFI-2 peptide against FXIIa, the role of CHFI protease-binding loop stabilization by the third disulfide bond was studied in experiments with CHFI-1245, which is a full-size CHFI mutant with the third disulfide bridge eliminated. This mutant was also used as a control for the C29D replacement in the CHFI-2 peptide. CHFI-1245 exhibits reduced inhibitory activity against FXIIa and trypsin . The shortened mutants CHFI-1234 (iK = 1.0 ± 0.3 nm for FXIIa) and CHFI-2345 (iK = 1.0 ± 0.2 nm) retain the ability to inhibit FXIIa to the same extent as wild-type CHFI (iK = 1.0 ± 0.1 nm); this finding is consistent with previously reported results (iK against FXIIa (3.2 ± 0.4 nm), which is similar to of CHFI-1234, CHFI-12345, and CHFI-2345 at a 0.01 level of significance . Similar results were obtained in the trypsin inhibition assay . Together, these results suggest that the 11 N-terminal and 24 C-terminal amino acids of CHFI and the first and the fifth disulfide bonds are not involved in interactions with FXIIa and trypsin. Therefore, CHFI-234 is a minimally active CHFI fragment that is necessary for FXIIa inhibition, and CHFI-2 is an independent structural element that retains its inhibitory activity against trypsin and FXIa.Our further studies of the contact region in the interaction between CHFI and FXIIa suggest that either a region between CHFI residues Glytigation  and the tigation  that a m results . CHFI-23 CHFI-1235. The sh CHFI-1235. The shInteractions outside the FXIIa active site and the CHFI protease-binding loop contribute to the strength and the specificity of binding, because the isolated binding loop is unable to inhibit FXIIa but partially retains inhibitory activity against other proteases, such as trypsin and FXIa. These findings suggest that the mechanism of FXIIa inhibition by CHFI could deviate from the standard mechanism , with no


Pseudomonas aeruginosa and Staphylococcus aureus are commonly associated with hospital-acquired infections and are known to form biofilms. Ciprofloxacin (CIP), which is normally used to treat these infections, is seldom effective in killing cells in a biofilm. This is mostly due to slow or weak penetration of CIP to the core of biofilms. The problem is accentuated by the release of CIP below MIC  levels following a rapid (burst) release. The aim of this study was to develop a drug carrier that would keep CIP above MIC levels for an extended period. Ciprofloxacin was suspended into poly (PDLLA) and poly(ethylene oxide) (PEO), and electrospun into nanofibers (CIP-F). All of the CIP was released from the nanofibers within 2 h, which is typical of a burst release. However, 99% of P. aeruginosa PA01 cells and 91% of S. aureus Xen 30 cells (a methicillin-resistant strain) in biofilms were killed when exposed to CIP-F. CIP levels remained above MIC for 5 days, as shown by growth inhibition of the cells in vitro. The nanofibers were smooth in texture with no bead formation, as revealed by scanning electron and atomic force microscopy. A single vibration peak at 1632 cm-1, recorded with Fourier transform infrared spectroscopy, indicated that CIP remained in crystal form when incorporated into PDLLA: PEO. No abnormalities in the histology of MCF-12A breast epithelial cells were observed when exposed to CIP-F. This is the first report of the inhibition of biofilm formation by CIP released from PDLLA: PEO nanofibers. Pseudomonas aeruginosa and Staphylococcus aureus are amongst the most dominant pathogens isolated from nosocomial infections  was used to perform one-way analysis of variance (ANOVA). -1 against 1 x 106 CFU ml-1P. aeruginosa PA01 and 16 mg l-1 against 1 x 106 CFU ml-1S. aureus Xen 30.The MIC of CIP was 4 mg lNanofibers without CIP (CF) were slightly larger in diameter ester bonds, respectively. Vibration peaks recorded at 1277 cm−1 and 1185 cm−1  recorded within the first 24 h (P. aeruginosa PA01 recorded when incubated in the presence of CF and in the absence of nanofibers (control) were more-or-less similar for the first 6 h. Optical density increased rapidly thereafter in the control (from OD600 0.3 to 1.7) within the following 18 h, but increased less drastically in the presence of CF (from OD600 0.2 to 0.6) over the same period  was recorded in both these biofilms (10 3 CFU ml–1 to log 10 6 CFU ml–1) in biofilms exposed to CIP-F , but lower protein (5.8 μg ml–1) concentrations were recorded for biofilm not exposed to nanofibers (control). No EPS, nor proteins, were detected in biofilms exposed to CIP-F. Similar results were recorded for S. aureus Xen 30 biofilms. Protein concentrations decreased from 10.3 μg ml–1 for the control to 4.9 μg ml–1 when exposed to CF, and to no detectible levels when exposed to CIP-F. No EPS production was detected in any of the S. aureus Xen 30 biofilms.The EPS and protein concentrations in No significant difference in cell viability was observed when breast epithelial cells MCF-12A were exposed to CF, CIP-F and in the absence of nanofibers . Almost -1  and those containing CIP (CIP-F), shown in -1 , which it in CIP . Detectit in CIP . No difft in CIP , withoutt in CIP , suggestP. aeruginosa PA01 in a liquid culture  to control cell growth for 5 days longer  and proteinaceous molecules of microorganisms play an important role in biofilm formation , 30. In The fact that no significant differences were recorded in the viability of MCF-12A breast cells exposed to CF, CIP-F and in the absence of nanofibers  indicateP. aeruginosa and S. aureus and safety against mammalian cells suggests that dressings produced from CIP-impregnated nanofibers may be used to prevent biofilm formation in topical infections caused by these bacteria.Ciprofloxacin was successfully encapsulated into PDLLA: PEO nanofibers. The amorphous distribution of CIP in nanofibers without noticeable chemical interactions, its ability to prevent biofilm formation by